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RESEARCH ARTICLE | MARCH 01 2017
Plant Lectins Activate the NLRP3 In ammasome To Promote In ammatory
Disorders 
Tao Gong; ... et. al
J Immunol (2017) 198 (5): 2082–2092.
https://doi.org/10.4049/jimmunol.1600145

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 The Journal of Immunology

Plant Lectins Activate the NLRP3 Inflammasome To Promote

Inflammatory Disorders

Tao Gong,* Xiaqiong Wang,* Yanqing Yang,* Yiqing Yan,* Chenggong Yu,†
Rongbin Zhou,*,‡,x,{ and Wei Jiang*,x
Plant-derived dietary lectins have been reported to be involved in the pathogenesis of several inflammatory diseases, including inflam-
matory bowel disease, diabetes, rheumatoid arthritis, and celiac disease, but little is known about the molecular mechanisms underlying
lectin-induced inflammation. In this study, we showed that plant lectins can induce caspase-1 activation and IL-1b secretion via the
NLRP3 inflammasome. Lectins were internalized and subsequently escaped from the lysosome and then translocated to the endoplasmic
reticulum. Endoplasmic reticulum–loaded plant lectins then triggered Ca2+ release and mitochondrial damage, and inhibition of Ca2+

Downloaded from http://journals.aai.org/jimmunol/article-pdf/198/5/2082/1428201/1600145.pdf by guest on 27 January 2024

release and mitochondrial reactive oxygen species by chemical inhibitors significantly suppressed NLRP3 inflammasome activation.
In vivo, plant lectin–induced inflammation and tissue damage also depended on the NLRP3 inflammasome. Our findings indicate that
plant lectins can act as an exogenous “danger signal” that can activate the NLRP3 inflammasome and suggest that dietary lectins might
promote inflammatory diseases via the NLRP3 inflammasome. The Journal of Immunology, 2017, 198: 2082–2092.

L
ectins are a heterogeneous group of proteins that were first
discovered in plants and subsequently in other species,
from microorganisms to humans. They share an important
biological propriety and specifically bind to carbohydrates in
a reversible way. This ability makes them act as a participator of
a wide range of biological events that are involved in protein–
carbohydrate recognition, such as cell development, cell recogni-
tion, tumor metastasis, host defense, and inflammation (1). Most
research efforts on lectins have been focused on plant lectins. Plant
*Institute of Immunology and the Chinese Academy of Sciences Key Laboratory of
Innate Immunity and Chronic Disease, Chinese Academy of Sciences Center for
Excellence in Molecular Cell Sciences, School of Life Sciences and Medical Center,
University of Science and Technology of China, Hefei 230027, China;
†
Department of Gastroenterology, Nanjing Drum Tower Hospital, Affiliated Hospital
of Nanjing University Medical School, Nanjing University, Nanjing 210008, China;
‡
Innovation Center for Cell Signaling Network, University of Science and Technol-
ogy of China, Hefei 230027, China; xHefei National Laboratory for Physical Sciences
at Microscale, University of Science and Technology of China, Hefei 230027, China;
and {State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell
Signaling Network, School of Life Sciences, Xiamen University, Xiamen 361101,
China
ORCIDs: 0000-0002-9414-6902 (T.G.); 0000-0001-9519-1732 (X.W.); 0000-0002-
3813-849X (Y. Yan); 0000-0001-8249-8321 (C.Y.).
Received for publication January 27, 2016. Accepted for publication December 19,
2016.
This work was supported by National Basic Research Program of China Grants
2013CB944904 and 2014CB910800, National Natural Science Foundation of China
Grants 31200659, 81273318, 81571609, 31300745, and 81525013, and by the Young
Talent Support Program and Fundamental Research Funds for the Central Universi-
ties (to R.Z. and W.J.).
Address correspondence and reprint requests to Prof. Wei Jiang, Hefei National
Laboratory for Physical Sciences at Microscale, University of Science and Technol-
ogy of China, Hefei 230027, China. E-mail address: ustcjw@ustc.edu.cn
The online version of this article contains supplemental material.
Abbreviations used in this article: ALT, alanine aminotransferase; Alum, aluminum
hydroxide; 2-APB, 2-aminoethoxydiphenyl borate; BiP, IgH-binding protein; BMDM,
bone marrow–derived macrophage; DAMP, danger-associated molecular pattern; ER,
endoplasmic reticulum; FAK, focal adhesion kinase; LCA, Lens culinaris agglutinin;
MBL, mannose-binding lectin; MnTBAP, manganese (III) tetrakis (4-benzoic acid)
porphyrin chloride; MSU, monosodium urate; PAMP, pathogen-associated molecular
pattern; poly(dA:dT), poly(deoxyadenylic-thymidylic) acid; ROS, reactive oxygen spe-
cies; SBA, soybean agglutinin; siRNA, small interfering RNA; WGA, wheat germ
agglutinin.
Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00
lectins are distributed in various kinds of foods, such as grains,
legumes, fruits, vegetables, and nuts (2). For some plants, they
make up as much as 10% of the total nitrogen in mature seed
extracts. Most importantly, plant lectins are more resistant to heat
denaturation and digestion than the animal proteins (3). Dietary
lectins can increase intestinal permeability and allow for the in-
creased translocation of both dietary and microbial Ags to the
periphery. Peripheral circulatory lectins not only provoke IgG and
IgM Ab production, but they also bind to cell surface glycopro-
teins, such as epidermal growth factor receptor and insulin re-
ceptor, and disrupt their functions (4–6). Therefore, lectins may
contribute to the pathogenesis of food intolerance, food allergy,
and other inflammatory diseases, such as inflammatory bowel
disease, insulin-dependent diabetes, rheumatoid arthritis, and IgA
nephropathy (6–9). Specifically, lectins from plants and microor-
ganisms have been considered as modulators of leukocyte func-
tion (10–13). Although the inflammatory effects of lectins are
emerging, the mechanisms are still poorly understood.
The innate immune system initiates inflammation and protects us
through pattern recognition receptors. Several members of pattern
recognition receptors act as sensor molecules, together with ASC
and procaspase-1, to form cytosolic protein complexes named the
“inflammasomes.” The inflammasomes are assembled in response
to microbial infection or “danger signals.” In contrast to NLRC4,
AIM2, NLRP1, and Pyrin inflammasomes, which are triggered by
a limited number of pathogen-associated molecular patterns
(PAMPs), the NLRP3 inflammasome not only can be activated
by several PAMPs, including viral RNA, bacterial surface protein,
and microbial toxins, but also by the widest array of danger-
associated molecular patterns (DAMPs), such as extracellular ATP,
monosodium urate (MSU), silica, aluminum adjuvant, hyaluronan,
and amyloid-b (14, 15). Owing to the diverse nature of PAMPs
and DAMPs that activate the NLRP3 inflammasome, how the
NLRP3 inflammasome is activated is not clearly understood.
The NLRP3 inflammasome is responsible for the maturation and
release of proinflammatory cytokines, such as IL-1b and IL-18,
and has been proposed to be involved in the pathogenesis of
various inflammatory diseases, including gout, diabetes, Alz-
heimer’s disease, atherosclerosis, and arthritis (14, 16). However,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1600145
The Journal of Immunology 2083

whether the NLRP3 inflammasome contributes to diet-induced
inflammatory diseases is still unclear.
In this study we found that plant lectins activated the NLRP3
inflammasome through the endoplasmic reticulum (ER) stress–
mitochondira axis. Furthermore, plant lectins induced NLRP3
inflammasome–dependent proinflammatory cytokine release and
tissue damage in vivo, suggesting that the NLRP3 inflammasome
may contribute to diet-induced inflammatory diseases.
Materials and Methods
Mice
Nlrp32/2, Asc2/2, Caspase-12/2, Ipaf2/2, Nod22/2, and Caspase-22/2
mice have been described (17–20). Nlrp62/2 and Nod12/2 mice were
provided by Millennium Pharmaceuticals. Nod12/2 and Nod22/2 mice
were crossed to generate Nod1/22/2 double-knockout mice. Itgal2/2 mice
were from The Jackson Laboratory. All mice were in a C57BL/6 back-
ground. The age- and sex-matched wild-type mice were used as controls.
Cell preparation and stimulation
Human THP-1 cells were obtained from American Type Culture Col-
lection. THP-1 cells were grown in RPMI 1640 medium plus 10% (v/v)
FBS and 50 mM 2-ME. THP-1 cells were treated for 3 h with 100 nM
PMA and then incubated overnight to differentiate into macrophages.
Bone marrow–derived macrophages (BMDMs) were derived from tibia
and femoral bone marrow cells as described and were cultured in DMEM
supplemented with 10% FBS, in the presence of L929 cell culture su-
pernatants (21).
For induction of inflammasome activation, 6 3 105 macrophages were
plated overnight in 12-well plates and then the cells were primed for 3 h
(using Opti-MEM supplemented with 1% FBS and 100 ng/ml ultrapure
LPS). After that, the cells were stimulated with various lectins, including
MSU (300 mg/ml) and Alum (300 mg/ml) for 6 h, or stimulated with ATP
(1 mM) and nigericin (5 mM) for 45 min. For poly(dA:dT) transfection, poly
(dA:dT) (1 mg/ml) was transfected by using Lipofectamine 2000 per the
manufacturer’s protocol (Invitrogen). Precipitated culture supernatants and
cell extracts were analyzed by Western blotting.

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All animal procedures were approved by the Ethics Committee of the
University of Science and Technology of China.
Reagents
Lectin from Canavalia ensiformis (Con A), Lens culinaris (L. culinaris ag-
glutinin [LCA]), Triticum vulgaris (wheat germ agglutinin [WGA]), Glycine
max (soybean agglutinin [SBA]), Arachis hypogaea (peanut agglutinin),
Ulex europaeus (U. europaeus agglutinin), Artocarpus integrifolia (jacalin),
nigericin, MSU, aluminum hydroxide (Alum), ATP, FITC-labeled Con A, FITC-
labeled WGA, bafilomycin A1, brefeldin A, 2-aminoethoxydiphenyl borate
(2-APB), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride
(MnTBAP), and PMA were from Sigma-Aldrich. PF-431396 and CA-074-Me
were from Selleck Chemicals. Ultrapure LPS, poly(deoxyadenylic-thymidylic)
acid [poly(dA:dT)], MitoTracker Red, LysoTracker Red, and MitoSOX Red
were from InvivoGen. Ab to IgH-binding protein (BiP) were from Proteintech.
Ab to mouse IL-1b was from R&D Systems. Abs to mouse caspase-1 (p20)
was from Adipogen International. Anti-calreticulin was from Abcam. Ab to
human IL-1b was from Sangon Biotech. Anti-human caspase-1 was from Cell
Signaling Technology.
Small interfering RNA–mediated gene silencing in THP-1 cells
PMA-differentiated THP-1 cells were plated in 12-well plates (at a density
of 6 3 105 cells per well) and then were transfected with 50 nM negative
control small interfering RNA (siRNA) or 25 nM of two different ITGAL
(encodes LFA-1a) siRNAs by using Lipofectamine RNAiMAX transfec-
tion reagent according to the manufacturer’s instructions (Invitrogen). The
siRNA sequence are as follows: 59-CGCCAATGTGACCTGTAACAA-39
and 59-CCCATCAATGTTTCCCTGAAT-39. All siRNA was chemically
synthesized by GenePharma.
ELISA
Mouse IL-1b, IL-6 (R&D Systems), and IL-18 (eBioscience) in superna-
tants of cell culture or peritoneal lavage fluid were quantified by ELISA
according to the manufacturers’ guidelines.
Confocal microscopy
THP-1 cells or BMDMs plated on coverslips (12-well plates, 2 3 105)
overnight were used for stimulation or staining with MitoTracker Red

FIGURE 1. Plant lectins induce caspase-1 activation and IL-1b secretion. (A) ELISA of IL-1b in culture supernatants from LPS-primed BMDMs
stimulated with different kinds of plant lectins for 6 h. (B and C) Immunoblot analysis of cleaved IL-1b and caspase-1 (p20) in culture supernatants (SN)
and inactive precursor molecule (pro–IL-1b, pro–caspase-1) in cell lysates (Input) of LPS-primed BMDMs stimulated with different doses of Con A (B) or
WGA (C) for 6 h. (D and E) Immunoblot analysis of cleaved IL-1b and caspase-1 (p20) in culture supernatants (SN) and inactive precursor molecule (pro–
IL-1b, pro–caspase-1) in cell lysates (Input) of PMA-differentiated THP-1 cells stimulated with different doses of Con A (D) or WGA (E) for 6 h. Data are
shown as mean 6 SEM values and represent three independent experiments. **p , 0.01, ***p , 0.001.
2084 PLANT LECTINS ACTIVATE THE NLRP3 INFLAMMASOME

(50 nM), LysoTracker Red (50 nM), or MitoSOX Red (1 mM) for 30 min.
After being washed twice, the cells were fixed for 20 min with 4% para-
formaldehyde and then were rinsed in PBST. For Ab staining, the cells
were permeabilized with 0.1% Triton X-100, blocked with 10% goat se-
rum in PBST, and then incubated overnight at 4˚C with anti-calreticulin in
10% goat serum in PBST. After being washed with PBST, cells were in-
cubated for 1 h with Alexa Fluor 546–conjugated secondary Ab (Invi-
trogen) in 10% goat serum in PBST and then were washed three times with
PBST. Nuclei were stained with DAPI (Invitrogen).
Real-time PCR
BMDM extraction was performed using TRIzol reagent (Invitrogen), and
cDNA was synthesized with the PrimeScript RT reagent kit (Takara Bio).
Quantitative real-time PCR was performed by AB StepOne real-time PCR
system (Applied Biosystems) and using SYBR Premix Ex TaqTM II (Takara
Bio). The primer sequences are as follows: BiP, 59-TCATCGGACG-
CACTTGGAA-39 (sense) and 59-CAACCACCTTGAATGGCAAGA-39
(antisense); Gapdh, 59-GGTGAAGGTCGGTGTGAACG-39 (sense) and
59-CTCGCTCCTGGAAGATGGTG-39 (antisense). The expression levels
enzymatic assay kit. Liver specimens were collected at 24 h and fixed with 4%
paraformaldehyde for 1 d and then embedded in paraffin. Paraffin-embedded
liver sections were cut into 4-mm slices, and all slices were deparaffinized prior
to staining with H&E.
WGA-induced cytokine release in vivo
Wild-type and NLRP3-deficient mice were injected i.p. with WGA (10 mg/kg,
dissolved in PBS). Six hours later, mice were sacrificed by exposure to CO2
and then peritoneal cavities were washed with 1 ml of cold PBS and the
concentrations of IL-1b, IL-18, or IL-6 were determined in the supernatant
of peritoneal lavage fluid by ELISA or Western blotting.
Statistical analyses
The data are expressed as the mean 6 SEM. Statistical significance
analyses were performed with the t test for two groups or two-way
ANOVA (GraphPad Prism software) for multiple groups. A p value
,0.05 was considered significant.
Results

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of BiP were normalized to the housekeeping gene Gapdh (DCt). The re-
sults were calculated by the 22DDCt method.
Flow cytometry
LPS-primed THP-1 cells or BMDMs were stimulated with FITC-labeled
Con A or FITC-labeled WGA for 1 h. After being washed twice, the
cells were digested and resuspended in cold PBS solution containing 1%
FBS. Live cells were gated and the mean fluorescence intensity of FITC was
analyzed using FACSVerse flow cytometry (BD Immunocytometry Sys-
tems). Data were analyzed using FlowJo software (Tree Star).
Con A–induced acute hepatitis
Con A was dissolved in PBS and i.v. injected into wild-type and NLRP3-
deficient mice via the tail vein. For survival experiments, mice were
monitored for 72 h after Con A (25 mg/kg) injection. For serum alanine
aminotransferase (ALT) assay and H&E staining analysis, Con A (15 mg/kg)
was injected. Serum was collected at 8 h to measure the levels of ALT with the
Plant lectins induce caspase-1 activation and IL-1b secretion
in macrophages
To investigate whether lectins could induce IL-1b maturation,
LPS-primed BMDMs were stimulated with various kinds of plant
lectins, including Con A, LCA, WGA, SBA, peanut agglutinin,
Ulex europaeus agglutinin, and jacalin. Secretion of IL-1b was
indeed detected after treatment with Con A, SBA, WGA, and LCA
(Fig. 1A), which belong to mannose or N-acetylglucosamine–
binding lectins. Because IL-1b secretion is controlled by caspase-
1 activity, we then tested whether Con A or WGA could activate
caspase-1. Treatment of BMDMs with Con A or WGA induced
caspase-1 activation and IL-1b maturation in a dose-dependent
manner (Fig. 1B, 1C). These results were also confirmed in hu-
man THP-1 cells (Fig. 1D, 1E). Thus, our results indicate that

FIGURE 2. The NLRP3 inflammasome is required for plant lectin–induced IL-1b maturation. (A) Immunoblot analysis of cleaved IL-1b and caspase-1
(p20) in culture supernatants (SN) and inactive precursor molecule (pro–IL-1b, pro–caspase-1) in cell lysates (Input) of LPS-primed wild-type (WT), Asc2/2, or
Caspase-12/2 (Casp12/2) BMDMs stimulated with different doses of Con A for 6 h. (B) Immunoblot analysis of cleaved IL-1b and caspase-1 (p20) in
culture supernatants (SN) and inactive precursor molecule (pro–IL-1b, pro–caspase-1) in cell lysates (Input) of LPS-primed WT or Nlrp32/2 BMDMs
stimulated with different doses of Con A for 6 h. (C) ELISA of IL-1b in culture supernatants from LPS-primed WT, Nlrp32/2, Asc2/2, or Casp12/2
BMDMs stimulated with different doses of WGA for 6 h. (D) ELISA of IL-1b in culture supernatants from LPS-primed WT and Nlrp32/2 BMDMs
stimulated with different lectins (200 mg/ml) for 6 h. Data are shown as mean 6 SEM values and represent three independent experiments. ***p , 0.001.
The Journal of Immunology 2085

mannose or N-acetylglucosamine–binding plant lectins, especially
Con A or WGA, can induce caspase-1 activation and IL-1b se-
cretion in mouse and human macrophages.
Plant lectins activate the NLRP3 inflammasome
Next we explored the pathway responsible for caspase-1 activation
and IL-1b maturation. BMDMs from Asc2/2 or caspase-12/2 mice
failed to release IL-1b after stimulation with Con A (Fig. 2A),
suggesting that lectin-induced IL-1b production depends on
inflammasomes. Next we sought to determine which inflammasome
was involved in the lectin-induced IL-1b release. The NLRP3
inflammasome is well conserved in mice and humans and can be
activated by various DAMPs. Con A could induce caspase-1 acti-
vation and IL-1b in wild-type BMDMs, but could not in NLRP3-
deficient cells (Fig. 2B). Similar to Con A, WGA-induced IL-1b
release was totally dependent on NLRP3, ASC, and caspase-1
Plant lectin Con A–induced NLRP3 inflammasome activation
in human macrophages depends on LFA-1
We next investigated the mechanism involved in lectin-induced
NLRP3 inflammasome activation. Lectins bind to numerous
carbohydrate-containing receptors enriched on the cell surface,
which may trigger cell signal transduction and physiological re-
sponses. For example, FimH, a bacteria lectin, can be recognized by
gp2, which is specifically expressed on the apical plasma mem-
brane of M cells among enterocytes and initiates mucosal immune
response (22). The cell surface receptors for Con A and WGA still
remain elusive. Integrin aLb2 (also called LFA-1) is a leukocyte
surface glycoprotein composed of a and b subunits. A previous
study has shown that LFA-1 binds to mammalian lectin Galectin-8
(23), and we therefore investigated whether LFA-1–mediated
plant lectins induced NLRP3 inflammasome activation. Con A
colocalized with LFA-1a in plasma membrane when THP-1 cells

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(Fig. 2C). LCA- or SBA-induced IL-1b release was also depen-
dent on the NLRP3 inflammasome (Fig. 2D). Other Nod-like re-
ceptors were not involved in IL-1b maturation induced by Con A or
WGA (Supplemental Fig. 1). Taken together, these results indicate
that plant lectins activate the NLRP3 inflammasome, which then
promotes caspase-1 activation and subsequent maturation of IL-1b.
were incubated with FITC–Con A (Fig. 3A). Inhibition of LFA-1a
expression by small interfering RNA (siRNA) significantly sup-
pressed the binding of Con A with THP-1 cells and the subsequent
inflammasome activation (Fig. 3B–D). In contrast, inhibition of
LFA-1a expression had no effect on the binding of WGA with
THP-1 cells or WGA-induced inflammasome activation (Fig. 3B–

FIGURE 3. Cell surface receptor is required for NLRP3 inflammasome activation by lectins. (A) Confocal microscopy analysis of PMA-differentiated
THP-1 cells stimulated with FITC-labeled Con A (10 mg/ml) for 1 h and then stained with anti–LFA-1a Ab. Scale bar, 20 mm. (B and C) PMA-differentiated
THP-1 cells were transfected with control siRNA or ITGAL (encodes LFA-1a)-specific siRNA for 48 h. siRNA-transfected cells were primed with LPS and
then stimulated with Con A (30 mg/ml) or WGA (10 mg/ml) for 6 h. (B) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants (SN) and
inactive precursor of caspase-1 (pro–caspase-1) in cell lysates (Input). (C) ELISA of IL-1b in culture supernatants. (D) PMA-differentiated THP-1 cells were
transfected with control siRNA or ITGAL-specific siRNA for 48 h. FACS analysis is shown of siRNA-transfected cells primed with LPS and then stimulated
with FITC-labeled Con A (10 mg/ml) or FITC-labeled WGA (5 mg/ml) for 1 h. (E and F) PMA-differentiated THP-1 cells were pretreated for 0.5 h with FAK
inhibitor (PF-431396, 10 mM) and then stimulated with Con A (30 mg/ml) or WGA (10 mg/ml) for 6 h. (E) Immunoblot analysis of cleaved caspase-1 (p20) in
culture supernatants (SN) and inactive precursor of caspase-1 (pro–caspase-1) in cell lysates (Input). (F) ELISA of IL-1b in culture supernatants. Data are
shown as mean 6 SEM values and represent three independent experiments. ***p , 0.001.
2086 PLANT LECTINS ACTIVATE THE NLRP3 INFLAMMASOME

D). Additionally, we found that Con A, but not WGA, treatment
resulted in the degradation of LFA-1a (Fig. 3B), suggesting that
Con A binding might promote the internalization and degradation
of LFA-1. However, Con A– or WGA-induced NLRP3 inflam-
masome activation or the binding of Con A or WGA to BMDMs
was normal in Itgal2/2 BMDMs, indicating that the mouse LFA-1
receptor is not involved in Con A–induced NLRP3 inflammasome
activation (Supplemental Fig. 2A, 2B). Ligand binding of the
integrin receptor promotes clustering of the integrin and subse-
quent recruitment of focal adhesion kinase (FAK) (24). We asked
whether FAK was involved in lectin-induced inflammasome ac-
tivation. Indeed, we found that PF-431396, an inhibitor of FAK,
could not inhibit Con A, WGA, or other inflammasome agonist-
induced caspase-1 activation or IL-1b production in both human
THP-1 cells or mouse BMDMs (Fig. 3E, 3F, Supplemental Fig.
2C–E), suggesting that the downstream signaling of LFA-1 was
not required for lectin-induced NLRP3 inflammasome activation.
Confocal analysis indicated that intracellular FITC–Con A not
only aggregated in the perinuclear space, but also diffusely dis-
tributed in cytoplasm (Fig. 4A). Indeed, internalized integrins and
ligands can traffic to the late endosomes and lysosomes for deg-
radation or can detach with ligands and then return to the plasma
membrane (25). Consistent with this, staining for lysosomes with
LysoTracker Red revealed aggregated Con A colocalized with
lysosomes (Fig. 4B). Several crystal-induced lysosomal damage
and rupture can activate the NLRP3 inflammasome, which can be
blocked by inhibition of phagosomal acidification (26). To test
whether lysosome-localized Con A contributes to inflammasome
activation, we used bafilomycin A1 to inhibit the vacuolar H+
ATPase, which is required for the acidification. Pretreatment with
bafilomycin A1 before lectin stimulation did not inhibit, but en-
hanced, caspase-1 activation and IL-1b maturation (Fig. 4C, 4D).
Additionally, inhibition of cathepsin B had no effect on lectin-
induced NLRP3 inflammasome activation (Supplemental Fig.

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Taken together, these results suggest that plant lectin–induced
NLRP3 activation depends on different integrin receptors, but not
its downstream signaling.
Lysosomal degradation negatively regulates plant
lectin–induced NLRP3 inflammaosme activation
Because the downstream signaling of LFA-1 is not required
for lectin-induced inflammasome activation, we then investigated
how cytosolic lectins induce NLRP3 inflammasome activation.
3A, 3B). These results indicate that lysosome-located lectins are
not responsible for NLRP3 inflammasome activation and that ly-
sosomal degradation negatively regulates lectin-induced NLRP3
inflammaosme activation.
Involvement of ER stress and mitochondrial damage in plant
lectin–induced NLRP3 inflammasome activation
We next examined whether diffusely distributed lectins activated
the NLRP3 inflammasome. First, we found that the diffuse FITC–

FIGURE 4. Lysosomal degradation negatively regulates plant lectin–induced NLRP3 inflammaosme activation. (A) Confocal microscopy analysis of
LPS-primed BMDMs stimulated with FITC-labeled Con A (30 mg/ml) for 1 h. Scale bar, 20 mm. (B) Confocal microscopy analysis of LPS-primed
BMDMs stimulated with FITC-labeled Con A (30 mg/ml) for 1 h, followed by staining with LysoTracker Red (50 nM). Scale bar, 20 mm. (C and D) LPS-
primed BMDMs were pretreated for 0.5 h with lysosome inhibitor (bafilomycin A1, 200 nM) and then stimulated with Con A (80 mg/ml), WGA (20 mg/ml),
or Alum (300 mg/ml) for 6 h or ATP (1 mM) for 45 min. (C) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants (SN) and inactive
precursor of caspase-1 (pro–caspase-1) in cell lysates (Input). (D) ELISA of IL-1b in culture supernatants. Data are shown as mean 6 SEM values and
represent three independent experiments. ***p , 0.001.
The Journal of Immunology 2087

Con A was colocalized with calreticulin, a marker for ER
(Fig. 5A). Inhibition of lysosomal acidification by bafilomycin A1
resulted in fewer Con A aggregates and more Con A colocaliza-
tion with ER (Fig. 5B). Because bafilomycin A1 enhanced
caspase-1 activation and IL-1b maturation (Fig. 4C, 4D), we
speculated that inhibition of lysosomal acidification led to the
escape of lectins from lysosomes and then activated the NLRP3
inflammasome via an ER-dependent manner.
The accumulation of misfolded proteins in the ER lumen lead to
ER stress, which then promotes mitochondrial damage, mito-
chondrial reactive oxygen species (ROS) production, and NLRP3
inflammasome activation (27, 28). To test whether ER-located
lectins induced ER stress and inflammasome activation, we
measured the expression of BiP, a monitor of ER stress. The ex-
pression of BiP was increased after Con A or WGA stimulation in
BMDMs (Fig. 5C, Supplemental Fig. 3C), suggesting that lectins

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FIGURE 5. ER-loaded plant lectins induce ER stress and mitochondrial damage to activate the NLRP3 inflammasome. (A) Confocal microscopy
analysis of LPS-primed BMDMs stimulated with FITC-labeled Con A (30 mg/ml) for 1 h and then stained with anti-calreticulin Ab. Scale bar, 20 mm. (B)
Confocal microscopy of LPS-primed BMDMs given no pretreatment (Mock) or pretreated for 0.5 h with lysosome inhibitor (bafilomycin A1, 200 nM),
followed by treatment and staining as in (A). Scale bar, 20 mm. (C) Immunoblot analysis of BiP in lysates of BMDMs stimulated with Con A (80 mg/ml),
WGA (20 mg/ml), or brefeldin A (BFA; 2 mg/ml) for 4 h. (D) Confocal microscopy of LPS-primed BMDMs left untreated or treated for 2 h with Con A
(30 mg/ml) or WGA (10 mg/ml) and then stained with mitochondrial dye MitoTracker Red (50 nM) and with the DNA-binding dye DAPI (blue). Scale
bar, 20 mm. (E) Confocal microscopy of LPS-primed BMDMs left untreated or treated for 4 h with Con A (30 mg/ml) or WGA (10 mg/ml) and then
stained with MitoSOX Red (1 mM) and with the DNA-binding dye DAPI (blue). Scale bar, 20 mm. (F and G) LPS-primed BMDMs were pretreated for
0.5 h with ROS inhibitor (MnTBAP, 150 mM) and then stimulated with Con A (80 mg/ml), WGA (20 mg/ml), or poly(dA:dT) (1 mg/ml) for 6 h or ATP
(1 mM) for 45 min. (F) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants (SN) and inactive precursor of caspase-1 (procaspase-1) in
cell lysates (Input). (G) ELISA of IL-1b in culture supernatants. Data are shown as mean 6 SEM values and represent three independent experiments.
***p , 0.001.
2088 PLANT LECTINS ACTIVATE THE NLRP3 INFLAMMASOME

induced ER stress in macrophages. Furthermore, treatment with
Con A or WGA promoted mitochondrial fission and aggregate
formation in the perinuclear space of BMDMs (Fig. 5D). Mito-
chondrial damage was accompanied by mitochondrial ROS pro-
duction (Fig. 5E). Inhibition of ROS produced by damaged
mitochondria with chemical inhibitor diminished lectin-induced
caspase-1 activation and IL-1b maturation (Fig. 5F, 5G), sug-
gesting that mitochondrial damage and ROS production are in-
volved in lectin-induced inflammaosme activation. These results
suggest that ER-located lectins induce ER stress, which then
promotes mitochondrial damage and NLRP3 inflammasome ac-
tivation.
Inhibition of Ca2+ release by chemical inhibitor suppresses
lectin-induced mitochondrial damage and inflammasome
activation
We further investigated how ER stress promoted mitochondrial
2–deficient BMDMs (Supplemental Fig. 4A), suggesting that
caspase-2 is not involved in lectin-induced inflammasome acti-
vation. We next investigated whether Ca2+ release from the
ER was responsible for the damage of mitochondria and subse-
quent inflammasome activation. We found that inhibition of Ca2+
release from the ER by 2-APB, the IP3 receptor antagonist, pre-
vented mitochondrial damage and ROS production (Fig. 6A,
Supplemental Fig. 4B). Moreover, 2-APB blocked lectin-induced
caspase-1 processing and IL-1b release in a dose-dependent
manner (Fig. 6B–E). Additionally, similar to ER stress-induced
NLRP3 inflammasome activation, lectin-induced NLRP3 inflam-
masome activation could be suppressed by extracellular potassium
(Supplemental Fig. 4C). Taken together, our results suggest that
Ca2+ signaling is involved in lectin-induced mitochondrial damage
and inflammasome activation.
Plant lectin-induced NLRP3 inflammasome activation

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damage and inflammasome activation. Published reports have
suggested that ER stress may promote mitochondrial damage
through different signal pathways. One study suggested ER stress
induces activation of caspase-2 and the proapoptotic factor Bid, and
then triggers mitochondrial dysfunction and NLRP3 inflammasome
activation (28). An alternative model is that Ca2+ release from
the ER mediates mitochondrial damage and amplifies NLRP3
inflammasome activation (29). To assess the role of caspase-2 in
lectin-induced NLRP3 inflammasome activation, we treated
BMDMs from caspase-2–deficient mice with Con A or WGA and
found that IL-1b release was similar in wild-type and caspase-
promotes inflammation in vivo
Abundant evidence indicates that plant lectins cause or contribute
to the current epidemic of chronic inflammatory illness and au-
toimmune disease (30, 31). We then asked whether the NLRP3
inflammasome was involved in lectin-induced inflammation
in vivo. Con A–induced acute liver injury is a well-known mouse
model of hepatitis. In this model, Th cells as well as macrophages
are the major effector cells (32). To test whether the NLRP3
inflammasome contributed to this pathological process, we chal-
lenged wild-type and NLRP3-deficient mice with Con A. Survival
analysis suggested that NLRP3-deficient mice exhibited delayed

FIGURE 6. ER-loaded plant lectins induce ER stress and mitochondrial damage to activate the NLRP3 inflammasome through Ca2+ signaling. (A)
Confocal microscopy of LPS-primed BMDMs given no pretreatment (Mock) or pretreated for 0.5 h with IP3R inhibitor (2-APB, 50 mM), followed by
treatment with Con A (30 mg/ml) for 2 h and then staining with the mitochondrial dye MitoTracker Red (50 nM) and with the DNA-binding dye DAPI
(blue). Scale bar, 20 mm. (B and C) LPS-primed BMDMs were pretreated for 0.5 h with various doses of 2-APB and then stimulated with Con A (80 mg/ml)
for 6 h. (B) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants (SN) and inactive precursor of caspase-1 (pro–caspase-1) in cell lysates
(Input). (C) ELISA of IL-1b in culture supernatants. (D and E) LPS-primed BMDMs were pretreated for 0.5 h with various doses of IP3R inhibitor (2-APB)
and then stimulated with WGA (20 mg/ml) for 6 h. (D) Immunoblot analysis of cleaved caspase-1 (p20) in culture supernatants (SN) and inactive precursor
of caspase-1 (pro–caspase-1) in cell lysates (Input). (E) ELISA of IL-1b in culture supernatants. Data are shown as mean 6 SEM values and represent three
independent experiments. **p , 0.01, ***p , 0.001.
The Journal of Immunology 2089

fatal injury (Fig. 7A). NLRP3-deficient mice also had diminished
serum ALT and reduced hepatic injury after Con A injection
(Fig. 7B, 7C). Consistent with this, the IL-1b levels in serum or
liver tissue were also compromised (Fig. 7D, 7E). These results
indicate that Con A–induced NLRP3 inflammasome activation
contributes to Con A–induced acute liver injury.
Among the food lectins, WGA is the most common one, which is
found in wheat germ (up to 0.5 g/kg) (33). Wheat is the staple food
for .35% of the global population (34). Published studies have
suggested that WGA increases intestinal permeability (35). In the
serum of healthy individuals, Abs to WGA have been found (36).
Significantly higher Ab levels to WGA were detected in patients
with celiac disease, and WGA may be involved in the pathogen-
esis of this disease (37). When wild-type and NLRP3-deficient
mice were i.p. injected with WGA, the production of IL-1b and
IL-18 in the peritoneal lavage fluid was impaired in NLRP3-
deficient mice (Fig. 8A–C). However, the production of IL-6,
that the NLRP3 inflammasome might play an important role in
diet-induced inflammatory disorders.
Lectins have been reported to bind to numerous carbohydrate-
containing receptors enriched on the cell surface. We identified
human LFA-1, a glycoprotein that belongs to integrin families, as
the receptor for Con A. LFA-1 was responsible for the binding and
internalization of Con A in human macrophages. Knockdown of
LFA-1a significantly suppressed Con A–induced inflammasome
activation, whereas it had no effect on WGA-induced inflamma-
some activation in human macrophage. Additionally, we found
that LFA-1 was not involved in Con A– or WGA-induced NLRP3
infalmmaosme activation in mouse BMDMs. One explanation for
this is that human or murine LFA-1a has a different affinity for
binding with Con A. Although human LFA-1a shares 72% of its
amino acids with its murine counterpart, human ICAM-1 can bind
to human, but not murine, LFA-1a, suggesting the existence of
species differences between mice and humans for this protein (38,

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which is released independent of the inflammasome, was similar
in wild-type and NLRP3-deficient mice (Fig. 8D). Taken together,
these results indicate that plant lectins can induce NLRP3
inflammasome activation and promote inflammation in vivo.
Discussion
Food intolerances or allergies lead to chronic digestive and au-
toimmune diseases. One of the key causes contributing to food-
induced illness may be plant lectins that are present in all foods,
such as grain lectins, legume lectins, and dairy lectins. In this study,
we found that plant lectins acted as an exogenous “danger signal”
and activated the NLRP3 inflammasome. Thus, our results suggest
39). Another explanation is that other integrins compensate the
deficiency of LFA-1a in mouse macrophages. Indeed, Con A
can bind to several other integrins, including LFA-1b, Mac-1a,
and CD11c (40–42). The role of these intergrins or other Con
A–binding proteins in plant lectin–induced NLRP3 inflammasome
activation needs to be further investigated. We also found that
some lectins, including jacalin, could not activate inflammasome
in macrophages, and this might be due to the lack of receptors for
these lectins in BMDMs. However, we cannot exclude the possi-
bility that jacalin might activate inflammasome in intestine, be-
cause it can bind glycoproteins, such as mucins and IgA1, which
are abundant in intestinal barrier (43, 44).

FIGURE 7. NLRP3 inflammasome activation contributes to Con A–induced acute liver injury. (A) Wild-type (WT) or Nlrp32/2 mice were injected with
a lethal dose of Con A (25 mg/kg). Survival was monitored every 4 h. (B) Serum ALT levels of WT or Nlrp32/2 mice 8 h after Con A injection (15 mg/kg).
(C) Liver H&E staining of WT or Nlrp32/2 mice 24 h after Con A injection (15 mg/kg). Scale bar, 200 mm; original magnification, 3100. (D) ELISA of IL-
1b in the serum of WT or Nlrp32/2 mice 8 h after Con A injection (15 mg/kg). (E) WT or Nlrp32/2 mice were injected with Con A (15 mg/kg) for 8 h.
Liver was isolated and cultured for 24 h, and supernatants were analyzed by ELISA for IL-1b. Data are shown as mean 6 SEM of six to eight mice per
group and represent three independent experiments. **p , 0.01, ***p , 0.001.
2090
FIGURE 8. WGA induces NLRP3
inflammasome activation in vivo. (A
and B) ELISA of IL-1b (A) and IL-18
(B) in the peritoneal cavity fluid of
wild-type (WT) or Nlrp32/2 mice 6 h
after i.p. injection with WGA (10 mg/kg).
(C) Immunoblot analysis of cleaved
IL-1b and IL-18 in the peritoneal
cavity fluid of WT or Nlrp32/2 mice
6 h after i.p. injection with WGA
(10 mg/kg). (D) ELISA of IL-6 in
the peritoneal cavity fluid of WT or
Nlrp32/2 mice 6 h after i.p. injec-
tion with WGA (10 mg/kg). Data are
shown as mean 6 SEM of six to
eight mice per group and represent
three independent experiments. **p ,
0.01, ***p , 0.001.
Along with their receptors, internalized lectins traffic to the late
endosomes and lysosomes for degradation. Our data indicated that
internalized lectins colocalized with the lysosomes and ER. We
speculated that lectins could escape from lysosomes and bind to
ER. In line with this hypothesis, inhibition of lysosomal degra-
dation led to more lectins colocalized with the ER. However, how
lectins escape from lysosomes is still unknown. Because lectins are
fairly resistant to proteolytic degradation (45), the accumulation of
lectins in lysosomes might have resulted in the leakage of lectins
from lysosomes. A cluster of guanylate-binding proteins (GTPa-
ses), which can induce the lysis of the pathogen-containing vac-
uole and release bacteria into the cytosol (46), may be involved in
the escape of lectins. The exact mechanisms for the escape of
lectins from lysosomes need to be further investigated.
Our results demonstrate that the downstream signaling of LFA-1
is not required for lecin-induced NLRP3 inflammaosme activation.
Inhibition of FAK kinase could not suppress Con A–induced
inflammasome activation. Additionally, we found that inhibition
of FAK enhanced Con A–induced NLRP3 inflammasome activa-
tion. A possible reason for this is that FAK can control actin as-
sembly via the Arp2/3 complex (47), and therefore inhibition of
FAK would block the trafficking of the LFA-1/Con A complex to
lysosomes and promote the escape of Con A from endosomes to
activate the NLRP3 inflammasome.
Several reports have linked ER stress to NLRP3 inflammasome
activation. Bronner et al. (28) reported that ER stress triggers
mitochondrial dysfunction and inflammasome activation through
an IRE1a–NLRP3–caspase-2–Bid signal pathway. However, they
also found that some compounds induced ER stress and NLRP3
inflammasome activation independent this pathway. This suggests
PLANT LECTINS ACTIVATE THE NLRP3 INFLAMMASOME
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that other types of signaling may participate in ER stress-induced
NLRP3 inflammasome activation. Murakami et al. (29) reported
that Ca2+ released from the ER during ER stress promote mito-
chondrial damage and NLRP3 inflammasome activation. Multiple
NLRP3 inflammasome activators induce Ca2+ mobilization, such
as ATP, crystals, nigericin, and high levels of extracellular Ca2+.
Con A and WGA have also been reported to induce intracellular
Ca2+ mobilization in different cell types (48, 49). Our results
suggest that Ca2+ released from the ER is involved in the lectin-
induced NLRP3 inflammasome activation, which is consistent
with the latter model.
Lectins are widely distributed in almost all species, from mi-
croorganisms to humans. Our results showed that mannose or
N-acetylglucosamine–binding plant lectins activated the NLRP3
inflammasome. Whether mannose or N-acetylglucosamine–binding
lectins from other species, especially pathogens and humans,
could activate the NLRP3 inflammasome needs to be further in-
vestigated. Indeed, the mannose-binding lectin (MBL), which is
the most intensively studied human lectin, also can mediate ER
stress and promote mitochondrial damage in renal tubular cells
independent of complement activation (50). Clinical data indicate
that MBL levels are higher in serum of patients suffering from
autoimmune diseases, such as type 1 diabetes, rheumatic heart
disease, and systemic lupus erythematosus (51–53). These results
suggest that human MBL might have a proinflammatory role, but
the role of the NLRP3 inflammasome needs to be further inves-
tigated.
Although we demonstrate that plant lectins can induce strong
NLRP3 inflammasome activation in macrophages, the concen-
tration needed for NLRP3 activation in vitro is high. Indeed, the
The Journal of Immunology
physiological concentration of lectins should be much lower, be-
cause the intestinal barrier could prevent the entry of lectins to the
body (54). However, in patients with inflammatory bowel disease,
the concentrations of serum WGA Abs are much higher than
in healthy controls (37), suggesting that more lectins can enter
the body and trigger NLRP3-dependent inflammation when the
intestinal barrier is disrupted. Additionally, the physiological
concentration of lectins might trigger low-grade and persistent
NLRP3-dependent inflammation, which then contributes to chronic
inflammatory diseases.
Our results demonstrate that Con A–induced NLRP3 inflam-
masome activation contributes to Con A–induced hepatitis. We
found that Con A treatment in Nlrp32/2 mice resulted in less
severe liver injury. This result is not consistent with a previous
report by Deutsch et al. (55) in which they showed that NLRP3
deficiency had no effects on Con A–induced liver injury. The
differences might be caused by the different doses used in the
experiments. Although NLRP3 is mainly expressed on monocytes,
macrophages, and dendritic cells, and macrophages are critical for
Con A–induced hepatitis (56, 57), we cannot exclude the possible
contribution of NLRP3-dependent pyroptosis in hepatocytes to
Con A–induced hepatitis, because constitutive NLRP3 activation
in mutant NLRP3 knock-in mice can cause hepatocyte pyroptosis,
which contributes to severe liver injury and inflammation (58).
Collectively, our results demonstrate that plant lectins can acti-
vate the NLRP3 inflammasome and promote secretion of proin-
flammatory cytokines, which may contribute to food-associated
chronic inflammation and inflammatory diseases.
Acknowledgments
We thank Dr. Jurg Tschopp for providing Nlrp32/2mice, Dr. Vishva M.
Dixit for providing Asc2/2 and Ipaf2/2 mice, Dr. Richard A. Flavell for
providing Caspase-1/112/2 mice, Dr. Andreas Strasser for providing Cas-
pase-22/2 mice, and Millennium Pharmaceuticals for providing Nlrp62/2
and Nod12/2 mice.
Disclosures
The authors have no financial conflicts of interest.
References
1. Sharon, N. 2007. Lectins: carbohydrate-specific reagents and biological recog-
nition molecules. J. Biol. Chem. 282: 2753–2764.
2. Damme, E. J. M. V., W. J. Peumans, A. Barre, and P. Rougé. 1998. Plant lectins:
a composite of several distinct families of structurally and evolutionary related
proteins with diverse biological roles. Crit. Rev. Plant Sci. 17: 575–692.
3. Pusztai, A., and G. Grant. 1998. Assessment of lectin inactivation by heat and
digestion. Methods Mol. Med. 9: 505–514.
4. Fälth-Magnusson, K., and K. E. Magnusson. 1995. Elevated levels of serum
antibodies to the lectin wheat germ agglutinin in celiac children lend support to
the gluten-lectin theory of celiac disease. Pediatr. Allergy Immunol. 6: 98–102.
5. Lochner, N., F. Pittner, M. Wirth, and F. Gabor. 2003. Wheat germ agglutinin
binds to the epidermal growth factor receptor of artificial Caco-2 membranes as
detected by silver nanoparticle enhanced fluorescence. Pharm. Res. 20: 833–839.
6. Pusztai, A., S. W. Ewen, G. Grant, D. S. Brown, J. C. Stewart, W. J. Peumans,
E. J. Van Damme, and S. Bardocz. 1993. Antinutritive effects of wheat-germ
agglutinin and other N-acetylglucosamine-specific lectins. Br. J. Nutr. 70: 313–
321.
7. Vasconcelos, I. M., and J. T. Oliveira. 2004. Antinutritional properties of plant
lectins. Toxicon 44: 385–403.
8. Watzl, B., C. Neudecker, G. M. Hänsch, G. Rechkemmer, and B. L. Pool-Zobel.
2001. Dietary wheat germ agglutinin modulates ovalbumin-induced immune
responses in Brown Norway rats. Br. J. Nutr. 85: 483–490.
9. Preedy, V. R., and R. R. Watson, eds. 2003. Reviews in Food and Nutrition
Toxicity, Vol. 1. Taylor & Francis, Boca Raton, FL.
10. Panunto-Castelo, A., M. A. Souza, M. C. Roque-Barreira, and J. S. Silva. 2001.
KM+, a lectin from Artocarpus integrifolia, induces IL-12 p40 production by
macrophages and switches from type 2 to type 1 cell-mediated immunity against
Leishmania major antigens, resulting in BALB/c mice resistance to infection.
Glycobiology 11: 1035–1042.
11. Ivory, C. P., and K. Chadee. 2007. Activation of dendritic cells by the Gal-lectin
of Entamoeba histolytica drives Th1 responses in vitro and in vivo. Eur.
J. Immunol. 37: 385–394.
2091
12. Vanderlei, E. S., K. K. Patoilo, N. A. Lima, A. P. Lima, J. A. Rodrigues,
L. M. Silva, M. E. Lima, V. Lima, and N. M. Benevides. 2010. Antinociceptive
and anti-inflammatory activities of lectin from the marine green alga Caulerpa
cupressoides. Int. Immunopharmacol. 10: 1113–1118.
13. Pires, A. F., N. V. Rodrigues, P. M. G. Soares, Rde. A. Ribeiro, K. S. Aragão,
M. M. Marinho, M. T. da Silva, B. S. Cavada, and A. M. S. Assreuy. 2016. A
novel N-acetyl-glucosamine lectin of Lonchocarpus araripensis attenuates acute
cellular inflammation in mice. Inflamm. Res. 65: 43–52.
14. Martinon, F., A. Mayor, and J. Tschopp. 2009. The inflammasomes: guardians of
the body. Annu. Rev. Immunol. 27: 229–265.
15. Yamasaki, K., J. Muto, K. R. Taylor, A. L. Cogen, D. Audish, J. Bertin,
E. P. Grant, A. J. Coyle, A. Misaghi, H. M. Hoffman, and R. L. Gallo. 2009.
NLRP3/cryopyrin is necessary for interleukin-1b (IL-1b) release in response to
hyaluronan, an endogenous trigger of inflammation in response to injury. J. Biol.
Chem. 284: 12762–12771.
16. Davis, B. K., H. Wen, and J. P. Ting. 2011. The inflammasome NLRs in immunity,
inflammation, and associated diseases. Annu. Rev. Immunol. 29: 707–735.
17. Martinon, F., V. Pétrilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Gout-
associated uric acid crystals activate the NALP3 inflammasome. Nature 440:
237–241.
18. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee,
M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation of
Downloaded from http://journals.aai.org/jimmunol/article-pdf/198/5/2082/1428201/1600145.pdf by guest on 27 January 2024
the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218.
19. Kobayashi, K. S., M. Chamaillard, Y. Ogura, O. Henegariu, N. Inohara,
G. Nuñez, and R. A. Flavell. 2005. Nod2-dependent regulation of innate and
adaptive immunity in the intestinal tract. Science 307: 731–734.
20. O’Reilly, L. A., P. Ekert, N. Harvey, V. Marsden, L. Cullen, D. L. Vaux,
G. Hacker, C. Magnusson, M. Pakusch, F. Cecconi, et al. 2002. Caspase-2 is not
required for thymocyte or neuronal apoptosis even though cleavage of caspase-2
is dependent on both Apaf-1 and caspase-9. Cell Death Differ. 9: 832–841.
21. Yan, Y., W. Jiang, T. Spinetti, A. Tardivel, R. Castillo, C. Bourquin, G. Guarda,
Z. Tian, J. Tschopp, and R. Zhou. 2013. Omega-3 fatty acids prevent inflam-
mation and metabolic disorder through inhibition of NLRP3 inflammasome
activation. Immunity 38: 1154–1163.
22. Hase, K., K. Kawano, T. Nochi, G. S. Pontes, S. Fukuda, M. Ebisawa,
K. Kadokura, T. Tobe, Y. Fujimura, S. Kawano, et al. 2009. Uptake through
glycoprotein 2 of FimH+ bacteria by M cells initiates mucosal immune response.
Nature 462: 226–230.
23. Vicuña, L., E. Pardo, C. Curkovic, R. Döger, C. Oyanadel, C. Metz, L. Massardo,
A. González, and A. Soza. 2013. Galectin-8 binds to LFA-1, blocks its inter-
action with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated
from lupus patients. Biol. Res. 46: 275–280.
24. McLean, G. W., N. O. Carragher, E. Avizienyte, J. Evans, V. G. Brunton, and
M. C. Frame. 2005. The role of focal-adhesion kinase in cancer—a new thera-
peutic opportunity. Nat. Rev. Cancer 5: 505–515.
25. Bouvard, D., J. Pouwels, N. De Franceschi, and J. Ivaska. 2013. Integrin inac-
tivators: balancing cellular functions in vitro and in vivo. Nat. Rev. Mol. Cell
Biol. 14: 430–442.
26. Hornung, V., F. Bauernfeind, A. Halle, E. O. Samstad, H. Kono, K. L. Rock,
K. A. Fitzgerald, and E. Latz. 2008. Silica crystals and aluminum salts activate
the NALP3 inflammasome through phagosomal destabilization. Nat. Immunol. 9:
847–856.
27. Menu, P., A. Mayor, R. Zhou, A. Tardivel, H. Ichijo, K. Mori, and J. Tschopp.
2012. ER stress activates the NLRP3 inflammasome via an UPR-independent
pathway. Cell Death Dis. 3: e261.
28. Bronner, D. N., B. H. Abuaita, X. Chen, K. A. Fitzgerald, G. Nuñez, Y. He,
X. M. Yin, and M. X. O’Riordan. 2015. Endoplasmic reticulum stress activates
the inflammasome via NLRP3- and caspase-2-driven mitochondrial damage.
Immunity 43: 451–462.
29. Murakami, T., J. Ockinger, J. Yu, V. Byles, A. McColl, A. M. Hofer, and
T. Horng. 2012. Critical role for calcium mobilization in activation of the
NLRP3 inflammasome. Proc. Natl. Acad. Sci. USA 109: 11282–11287.
30. Hamid, R, and A Masood. 2009. Dietary lectins as disease causing toxicants.
Pak. J. Nutr. 8: 293–303.
31. Cordain, L., L. Toohey, M. J. Smith, and M. S. Hickey. 2000. Modulation of
immune function by dietary lectins in rheumatoid arthritis. Br. J. Nutr. 83: 207–
217.
32. Gantner, F., M. Leist, A. W. Lohse, P. G. Germann, and G. Tiegs. 1995. Con-
canavalin A-induced T-cell-mediated hepatic injury in mice: the role of tumor
necrosis factor. Hepatology 21: 190–198.
33. Peumans, W. J., and E. J. M. Van Damme. 1996. Prevalence, biological activity
and genetic manipulation of lectins in foods. Trends Food Sci. Technol. 7: 132–
138.
34. Williams, P. C. 1993. The World of Wheat, Grain and Oil Seeds. Vol. 2. 4th Ed.
Canadian International Grain Institute, Winnipeg, Canada, p. 578–590.
35. Dalla Pellegrina, C., O. Perbellini, M. T. Scupoli, C. Tomelleri, C. Zanetti,
G. Zoccatelli, M. Fusi, A. Peruffo, C. Rizzi, and R. Chignola. 2009. Effects of
wheat germ agglutinin on human gastrointestinal epithelium: insights from an
experimental model of immune/epithelial cell interaction. Toxicol. Appl. Phar-
macol. 237: 146–153.
36. Tchernychev, B., and M. Wilchek. 1996. Natural human antibodies to dietary
lectins. FEBS Lett. 397: 139–142.
37. Sollid, L. M., J. Kolberg, H. Scott, J. Ek, O. Fausa, and P. Brandtzaeg. 1986. Anti-
bodies to wheat germ agglutinin in coeliac disease. Clin. Exp. Immunol. 63: 95–100.
38. Kaufmann, Y., E. Tseng, and T. A. Springer. 1991. Cloning of the
murine lymphocyte function-associated molecule-1 alpha-subunit and its ex-
pression in COS cells. J. Immunol. 147: 369–374.
2092
39. Huang, C., and T. A. Springer. 1995. A binding interface on the I domain
of lymphocyte function-associated antigen-1 (LFA-1) required for specific in-
teraction with intercellular adhesion molecule 1 (ICAM-1). J. Biol. Chem. 270:
19008–19016.
40. Schmalstieg, F. C., H. E. Rudloff, and D. C. Anderson. 1986. Binding of the
adhesive protein complex (LFA-1/Mac-1/p150,95) to concanavalin A. J. Leukoc.
Biol. 39: 193–203.
41. Lacal, P. M., J. Balsinde, C. Cabañas, C. Bernabeu, F. Sánchez-Madrid, and
F. Mollinedo. 1990. The CD11c antigen couples concanavalin A binding to
generation of superoxide anion in human phagocytes. Biochem. J. 268: 707–712.
42. Sitkovsky, M. V., M. S. Pasternack, J. P. Lugo, J. R. Klein, and H. N. Eisen.
1984. Isolation and partial characterization of concanavalin A receptors on
cloned cytotoxic T lymphocytes. Proc. Natl. Acad. Sci. USA 81: 1519–1523.
43. Loomes, L. M., W. W. Stewart, R. L. Mazengera, B. W. Senior, and M. A. Kerr.
1991. Purification and characterization of human immunoglobulin IgA1 and
IgA2 isotypes from serum. J. Immunol. Methods 141: 209–218.
44. Saroha, A., S. Kumar, B. P. Chatterjee, and H. R. Das. 2012. Jacalin bound
plasma O-glycoproteome and reduced sialylation of alpha 2-HS glycoprotein
(A2HSG) in rheumatoid arthritis patients. PLoS One 7: e46374.
45. Clement, F., and Y. P. Venkatesh. 2010. Dietary garlic (Allium sativum) lectins,
ASA I and ASA II, are highly stable and immunogenic. Int. Immunopharmacol.
10: 1161–1169.
46. Meunier, E., M. S. Dick, R. F. Dreier, N. Sch€urmann, D. Kenzelmann Broz,
S. Warming, M. Roose-Girma, D. Bumann, N. Kayagaki, K. Takeda, et al. 2014.
Caspase-11 activation requires lysis of pathogen-containing vacuoles by IFN-
induced GTPases. Nature 509: 366–370.
47. Serrels, B., A. Serrels, V. G. Brunton, M. Holt, G. W. McLean, C. H. Gray,
G. E. Jones, and M. C. Frame. 2007. Focal adhesion kinase controls actin as-
sembly via a FERM-mediated interaction with the Arp2/3 complex. Nat. Cell
Biol. 9: 1046–1056.
48. Grinstein, S., J. D. Smith, C. Rowatt, and S. J. Dixon. 1987. Mechanism of
activation of lymphocyte Na+/H+ exchange by concanavalin A. A calcium- and
protein kinase C-independent pathway. J. Biol. Chem. 262: 15277–15284.
49. Yatomi, Y., Y. Ozaki, Y. Koike, K. Satoh, and S. Kume. 1993. Wheat germ
agglutinin-induced intracellular calcium mobilization in human platelets: sup-
PLANT LECTINS ACTIVATE THE NLRP3 INFLAMMASOME
pression by staurosporine and resistance to cyclic AMP inhibition. Biochem.
Biophys. Res. Commun. 191: 453–458.
50. Pieter van der, P. 2013. Pathogenic Role of Complement in Renal Ischemia/
Reperfusion Injury. Department of Nephrology, Faculty of Medicine/Leiden
University Medical Center, Leiden University, Leiden, the Netherlands.
51. Hansen, T. K., S. Thiel, S. T. Knudsen, C. H. Gravholt, J. S. Christiansen,
C. E. Mogensen, and P. L. Poulsen. 2003. Elevated levels of mannan-binding
lectin in patients with type 1 diabetes. J. Clin. Endocrinol. Metab. 88: 4857–
4861.
52. Schafranski, M. D., A. Stier, R. Nisihara, and I. J. T. Messias-Reason. 2004.
Significantly increased levels of mannose-binding lectin (MBL) in rheumatic
heart disease: a beneficial role for MBL deficiency. Clin. Exp. Immunol. 138:
521–525.
53. Panda, A. K., J. R. Parida, R. Tripathy, S. S. Pattanaik, B. Ravindran, and
B. K. Das. 2012. Mannose binding lectin: a biomarker of systemic lupus
erythematosus disease activity. Arthritis Res. Ther. 14: R218.
54. Mandal, C., and C. Mandal. 1990. Sialic acid binding lectins. Experientia 46:
433–441.
55. Deutsch, M., C. S. Graffeo, R. Rokosh, M. Pansari, A. Ochi, E. M. Levie, E. Van
Heerden, D. M. Tippens, S. Greco, R. Barilla, et al. 2015. Divergent effects of
RIP1 or RIP3 blockade in murine models of acute liver injury. Cell Death Dis. 6:
Downloaded from http://journals.aai.org/jimmunol/article-pdf/198/5/2082/1428201/1600145.pdf by guest on 27 January 2024
e1759.
56. Guarda, G., M. Zenger, A. S. Yazdi, K. Schroder, I. Ferrero, P. Menu,
A. Tardivel, C. Mattmann, and J. Tschopp. 2011. Differential expression of
NLRP3 among hematopoietic cells. J. Immunol. 186: 2529–2534.
57. Nakashima, H., M. Kinoshita, M. Nakashima, Y. Habu, S. Shono, T. Uchida,
N. Shinomiya, and S. Seki. 2008. Superoxide produced by Kupffer cells is an
essential effector in concanavalin A–induced hepatitis in mice. Hepatology 48:
1979–1988.
58. Wree, A., A. Eguchi, M. D. McGeough, C. A. Pena, C. D. Johnson, A. Canbay,
H. M. Hoffman, and A. E. Feldstein. 2014. NLRP3 inflammasome activa-
tion results in hepatocyte pyroptosis, liver inflammation, and fibrosis in mice.
Hepatology 59: 898–910.